PRELP Protein Inhibits the Formation of the Complement Membrane Attack Complex

PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins

The C-Type Lectin of the Aggrecan G3 Domain Activates Complement

Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint

Interactions between extracellular matrix proteins and the complement system - In the perspective of cartilage degradation in inflammatory joint diseases

Abstract: The joint diseases osteoarthritis and rheumatoid arthritis are characterized by destructive inflammatory processes that result in pathological changes of the joint tissues, including proteolytic degradation of cartilage and release of extracellular matrix proteins or fragments to the synovial fluid. The complement system, which is a part of the innate immune system, plays a central role in promoting the joint inflammation in these diseases. Potential activators of complement are certain extracellular matrix proteins that become exposed during cartilage degradation. Previous studies have revealed that several proteins from cartilage can activate or inhibit the complement system in vitro. In the present work, we describe the interactions between the complement system and additional extracellular matrix proteins, with the aim to better understand the role of endogenous ligands in the inflammatory process in joint diseases. We also describe the interactions between complement proteins and cartilage explants that have been subjected to inflammation-induced degradation. In paper I, we found that the G3 domain of aggrecan activates the classical pathway of complement. However, the activation of the terminal pathway is limited due to the simultaneous binding of factor H. Whether it activates complement when maintained in cartilage or when released into the synovial fluid remains to be elucidated. In paper II and III, we found that both proline/arginine-rich end leucine-rich repeat protein (PRELP) and the domain NC4 of collagen type IX, inhibit complement by preventing the assembly of the membrane attack complex. Further, PRELP also inhibits the assembly of the alternative pathway C3 convertase, while NC4 enhances the cofactor activities of C4b-binding protein and factor H, in the factor I-mediated cleavage of C4b and C3b. NC4 and fragments of PRELP can be detected in the synovial fluid of rheumatoid arthritis patients. Located in the synovial fluid or exposed on the cartilage surface, they might be important for limiting the complement activation, induced by other extracellular matrix proteins or other potential triggers. In Paper IV, we found that both the classical and the alternative pathways are activated on the surface of degraded cartilage explants, while components released from cartilage might have a weak, somewhat delayed, opposing role by inhibiting complement. The main activation seems to occur after the major loss of aggrecan from cartilage. In sum, several proteins of the extracellular matrix, as well as degraded cartilage have the potential to interact with the complement system, and may regulate the inflammatory processes in joint diseases

The NC4 domain of the cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H.

Collagen IX containing the N-terminal non-collagenous domain 4 (NC4) domain is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and non-collagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein (COMP), matrilins, fibromodulin and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3 and C9, and to directly inhibit C9 polymerisation and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together our results suggest that collagen IX and its interactions with matrix components is an important part of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis

Lectin domain of aggrecan G3 module activates the classical pathway.

<p>Aggregated IgG (positive control for classical pathway), LCt, Lt, Ct and BSA (negative control) were coated onto microtiter plates and increasing concentrations of normal human serum (NHS) were added. Activation of the classical pathway was measured by detecting deposited C1q (<b>A</b>), C4b (<b>B</b>), C3b (<b>C</b>) and C9 (<b>D</b>). The graphs show the mean and standard deviation (SD) of three independent experiments. Statistical significance of differences was calculated using a two-way ANOVA with a Bonferroni posttest. * p<0.05, **p<0.01, ***p<0.001.).</p

C1q binds the G3 domain and a part of the aggrecan core protein.

<p><b>A–E,</b> using electron microscopy, complexes between bovine nasal cartilage aggrecan and C1q were visualized by negative staining. G1 and G3 domains of aggrecan were labelled with antibody-conjugated colloidal gold (10 nm and 5 nm, respectively) and are pointed out by black (G1) and white (G3) arrowheads. Scale bar shows 100 nm (A) and 50 nm (B). <b>A</b>, several aggrecan molecules bound to the heads of C1q through their G3 domains. C1q also bound the core of aggrecan. <b>B–D</b>, selected complexes between single molecules of C1q and one to three aggrecan molecules through their G3 domains, at a higher magnification. <b>E</b>, single molecule of C1q bound to the core of aggrecan. <b>F</b>, microtiter plates were coated with aggregated IgG (positive control), adult, calf and fetal bovine articular cartilage aggrecan and BSA (negative control) and deposited C4b from increasing concentrations of NHS was detected. The graph show the mean and SD of three independent experiments. Statistical significance of differences was calculated using two-way ANOVA with Bonferroni posttest. (*p<0.05, **p<0.01, ***p<0.001.).</p

The mutation D2267N within the G3 domain decreases the complement activation capacity.

<p><b>A–B</b>, microtiter plates were coated with aggregated IgG (positive control for classical pathway), LCt carrying the mutation D2267N or V2303M, wt LCt, and BSA (negative control). <b>A</b>, increasing concentrations of NHS were added and complement activation was measured by detecting deposited C4b. <b>B</b>, plates were incubated with purified C1q, which was then detected with specific antibodies. <b>C</b>, Microtiter plates were coated with untreated or N-glycosidase F-treated D2267N LCt, aggregated IgG and BSA. Proteins were incubated with increasing concentrations of NHS and deposited C4b was detected. Note that in addition to deglycosylation, the deamidation resulting from N-glycosidase F treatment reverts the protein sequence to wild type. <b>D</b>, D2267N LCt was deglycosylated with N-glycosidase F and untreated and deglycosylated proteins were separated by SDS/PAGE followed by Coomassie Blue staining. The graphs show the mean and SD of three independent experiments. Statistical significance of differences was calculated using two-way ANOVA with Bonferroni posttest. (*p<0.05, **p<0.01, ***p<0.001.).</p

Lectin domain of aggrecan G3 domain activates classical pathway due to binding of C1q.

<p><b>A</b>, aggregated IgG (positive control for classical pathway), mannan (positive control for lectin pathway), LCt, Lt, Ct and BSA (negative control) were coated onto microtiter plates and increasing concentrations of C1q-depleted human serum were added. Activation of the classical pathway was measured by detecting deposited C4b. The absorbance was normalized relative to deposition on mannan from 10% C1q-depleted serum. <b>B</b>, aggregated IgG, LCt, Lt, Ct and BSA were coated onto microtiter plates and incubated with purified C1q, which was then detected with specific antibodies. Aggregated IgG, LCt, Lt, Ct and BSA were coated onto microtiter plates and incubated with purified C1q in a buffer supplemented with increasing NaCl concentrations (<b>C</b>) or EDTA (<b>D</b>). The graphs show the mean and SD of three independent experiments. Statistical significance of differences was calculated using two-way ANOVA with Bonferroni posttest (B, D). (*p<0.05, **p<0.01, ***p<0.001.).</p

Lectin domain of aggrecan G3 module binds factor H (FH).

<p>Microtiter plates were coated with fibromodulin (positive control for binding of FH), LCt, Lt, Ct and BSA (negative control) and the binding of FH from heat-inactivated NHS (hi-NHS) (<b>A</b>) or solution containing purified FH (<b>B</b>) was detected using specific antibodies. Fibromodulin, LCt, Lt, Ct and BSA were coated onto microtiter plates and incubated with purified FH in a buffer supplemented with increasing NaCl concentrations (<b>C</b>) or EDTA (<b>D</b>). In A, C and D absorbance was normalized relative to the highest absorbance value obtained with fibromodulin in each figure. The graphs show the mean and SD of three independent experiments. The differences in binding using different EDTA concentrations (D) were not statistically significant, calculated using two-way ANOVA with Bonferroni posttest. (*, p<0.05, **, p<0.01, ***, p<0.001.).</p

Structure of aggrecan and localization of G3 domain fragments which were used in the study.

<p><b>A</b>, schematic outline of human aggrecan, including three globular domains (G1–G3), which is involved in numerous interactions with other ECM components. The carboxyl-terminal globular G3 domain consists of one or two EGF-like domains, a C-type lectin domain (CLD) and a complement control protein (CCP) domain followed by a short tail. In the current study we used three wild-type recombinant fragments of G3 domain: LCt, Lt and Ct and two mutants of LCt containing D2267N or V2303M substitutions. <b>B</b>, the wild-type and mutated fragments of G3 domain were expressed in eukaryotic cells and separated by SDS/PAGE followed by Coomassie Blue staining.</p